This invention relates to a reagent for measurement of leukocytes and hemoglobin concentration in a blood sample.
Measuring the leukocyte count and hemoglobin concentration in the blood is very important for the clinical diagnosis of leukemia, anemia, etc. or for monitoring the clinical course in patients.
Currently, the leukocyte count and hemoglobin concentration can be measured in a short time by an automatic blood analyzer, and such an analyzer is in widespread use.
Automatic blood analyzers can be roughly classified into an optical detection system for detecting light scattering or fluorescence, and an electric resistance detection system for detecting a change in impedance occurring when particles pass through an aperture. The latter system is superior in terms of ease of use. According to the electric resistance detection system, the measurement of the leukocyte count is performed by adding a hemolytic agent to a blood sample to lyse erythrocytes and prepare a sample for leukocyte measurement with only leukocytes left, and then flowing this sample through a detector to detect a signal issued.
Normally, leukocytes in the peripheral blood include five types of cells, lymphocyte, monocyte, neutrophil, eosinophil and basophil. By formulating the hemolytic agent in a preferred manner, it becomes possible to classify the leukocytes into 2 or 3 populations, or detect only leukocytes of a particular type. Thus, leukocyte classification that has necessarily relied on microscopic observation can be performed automatically in a short time. This has reduced the burden on laboratory technicians, and has obviated the need for special technique for laboratory examination.
To measure hemoglobin concentrations, on the other hand, it has been adopted as the international standard method to convert hemoglobin into cyanmethemoglobin by the use of Van Kampen reagent containing a cyanogen compound, and measuring its absorbance at about 541 nm. For an automatic blood cell analyzer, a method is known which fragments erythrocytes, and denatures hemoglobin, with a quaternary ammonium salt having surface activity, obtains a cyanmethemoglobin-like absorption curve with the use of an alkali cyanide, and determines hemoglobin as well as leukocytes. However, harmful cyanogen compounds are contained in the reagents. These reagents or the samples after measurement need to be subsequently made atoxic before being discarded.
There is a known reagent which permits leukocyte counting without using a cyanogen compound for the determination of the hemoglobin concentration (U.S. Pat. No. 4,185,964). This reagent contains a water-soluble quaternary ammonium salt having surface activity, and a small amount of a polycarboxylic acid having up to about 8 carbon atoms in an amount sufficient to inhibit the lysis of leukocytes. Hemolytic agents which enable leukocytes to be classified into three populations are also known. These hemolytic agents contain quaternary ammonium salts and specific hemoglobin stabilizers (Japanese Unexamined Patent Publication Nos. 3-137566, 3-252557 and 4-13969).
The above hemolytic agents can denature hemoglobin rapidly, but have the drawback that the hemoglobin concentration markedly varies with the liquid temperature of the blood sample. Particularly in U.S. Pat. No. 4,185,964, the variations are great, since no stabilizer is used. In Japanese Unexamined Patent Publication Nos. 3-137566, 3-252557 and 4-13969, specific hemoglobin stabilizers are incorporated, so that the variations are reduced. However, when the liquid temperature fluctuates, the hemoglobin concentration also fluctuates. The stabilizing effect of these stabilizers is insufficient in this case. To obtain stable measurements, therefore, a unit for keeping the liquid temperature constant is required. This is one of the factors leading to an increase in the cost of the apparatus.
An object of the present invention is to provide a reagent which does not use a cyanogen compound, can measure the leukocyte count, and can also measure the hemoglobin concentration stably even when the liquid temperature of the sample varies.
The reagent for measurement of leukocytes and hemoglobin concentration according to the present invention contains:
(1) a cationic surfactant in an amount sufficient to lyse erythrocytes and denature hemoglobin,
(2) at least one hemoglobin stabilizer selected from the group consisting of the following (a), (b) and (c):
(a) sulfosalicylic acid, or its salt, in an amount effective for promoting the conversion of hemoglobin into methemoglobin,
(b) 0.2 to 10.0 g/L of a water-soluble chelating agent having a nitrogen atom and a carboxyl group, and
(c) piperazine, or its salt, in an amount effective for promoting the conversion of hemoglobin into methemoglobin, and
(3) a buffer for maintaining pH at 4 to 6.
The use of the reagent for measurement of leukocytes and hemoglobin concentration according to the present invention enables leukocytes and hemoglobin concentration to be measured simultaneously or separately.
The reagent for measurement of leukocytes and hemoglobin concentration according to the present invention contains a cationic surfactant in an amount sufficient to lyse erythrocytes and denature hemoglobin. The cationic surfactant preferably includes at least one cationic surfactant of the quaternary ammonium salt type or pyridinium salt type having the structure indicated below.
Preferred examples are as follows:
(a) Quaternary ammonium salts 
where R1 represents a C8-C20 alkyl group, alkenyl group or alkinyl group, R2, R3 and R4 represent, independently of each other, a C1-C8 alkyl group, alkenyl group or alkinyl group, and Xxe2x88x92 represents a halogen ion.
(b) Pyridinium salts 
where n denotes an integer of 7 to 19, and Xxe2x88x92 represents a halogen ion.
The preferred concentration of the cationic surfactant used in the reagent of the present invention is 0.1 to 15.0 g/L. The preferred cationic surfactant is a quaternary ammonium salt, and the particularly preferable concentration is 0.1 to 4.0 g/L. By suitably combining the surfactants stated above, leukocytes can be classified, for example, into lymphocytes and others, or into lymphocytes, neutrophils and others.
The reagent for measurement of leukocytes and hemoglobin concentration according to the present invention also contains a hemoglobin stabilizer in an amount effective for promoting the conversion of hemoglobin into methemoglobin. The stabilizer is at least one member selected from the group consisting of (a) sulfosalicylic acid or its salt, (b) a water-soluble chelating agent having a nitrogen atom and a carboxyl group, and (c) piperazine or its salt. Examples of the water-soluble chelating agent are ethylenediaminetetraacetic acid or its salts, diaminopropanoltetraacetic acid or its salts, diaminopropanetetraacetic acid or its salts, ethylenediaminediacetic acid or its salts, and ethylenediaminedipropionic acid or its salts. Particularly preferred are ethylenediaminetetraacetates.
The hemoglobin stabilizer is used in an amount effective for promoting the conversion of hemoglobin into methemoglobin. Normally, it is used in a preferred amount in the range of 0.2 to 10.0 g/L. A more preferable concentration differs depending on the substance used. When sulfosalicylic acid or its salt is used, the more preferable concentration is 0.2 to 2.0 g/L. When the water-soluble chelating agent is used, a particularly preferred concentration is 0.5 to 10 g/L for ethylenediaminetetraacetic acid salt, 0.5 to 5 g/L for diaminopropanoltetraacetic acid or its salt, 0.5 to 5 g/L for diaminopropanetetraacetic acid or its salt, 0.5 to 5 g/L for ethylenediaminediacetic acid or its salt, or 0.5 to 5 g/L for ethylenediaminedipropionic acid dihydrochloride. In the case of piperazine or its salt, it is used preferably in a concentration of 0.5 to 5 g/L. When this stabilizer has a buffer action, it may be used as part of the buffer.
These stabilizers are presumed to exhibit their stabilizing effect by binding to the heme of methemoglobin denatured with the aforementioned cationic surfactant or a combination of the cationic surfactants.
The buffer is not restricted as long as it can maintain a pH at 4.0 to 6.0. Too low a pH makes leukocytes fragile, thereby adversely affecting the measurement of the leukocyte count. Too high a pH, on the other hand, results in poor stability of hemoglobin over time. Concretely, known buffers can be used, such as Good buffer, phosphate buffer, succinate buffer, and maleate-TRIS buffer. The concentration of the buffer is 5 to 50 mM, preferably 15 to 30 mM.
The above-mentioned concentrations of the respective components are the concentrations used when the blood is directly diluted with the reagent for measurement of leukocytes and hemoglobin concentration according to the present invention. Alternatively, the reagent may be added after the blood is diluted with a diluent (e.g., physiological saline solution or Cellpack(trademark) (TOA MEDICAL ELECTRONICS CO., LTD.)). In this case, it suffices to adjust the concentrations of the respective components so that after addition of the reagent, these concentrations would fall into the indicated ranges. The above diluent is preferably one with a pH close to neutrality (6 to 8) and an osmotic pressure close to isotonicity (240 to 330 Osm/kg) for maintaining the shape of the blood cells.
The reagent of the present invention has electric conductivity, preferably, of 8 to 20 mS/cm, and more preferably, of 10 to 15 mS/cm. The electric conductivity can be adjusted by suitably adding an electrolyte such as NaCl.
The leukocyte count and the hemoglobin concentration can be measured in the following manner: A blood sample is prepared using the reagent for measurement of leukocytes and hemoglobin concentration according to the present invention. The blood sample is introduced into an automatic blood cell analyzer. In this apparatus, a change in impedance occurring during passage of particles through an aperture is detected to measure the leukocyte count, and the absorbance of the sample is detected to measure the hemoglobin concentration. In this case, the leukocyte count and the hemoglobin concentration can be measured simultaneously or separately.
Formulation examples of the reagent of the present invention are offered below.
With Formulation Examples 1 to 3, leukocyte and hemoglobin can be measured simultaneously. By adjusting the type and concentration of each surfactant, leukocytes can be classified into two or more populations. The populations of leukocytes refer, for example, to lymphocytes, monocytes, neutrophils, eosinophils and basophils. Formulation Example 1 is a combination of the components designed to classify leukocytes into a single population, while Formulation Examples 2 and 3 are combinations of the components designed to classify leukocytes into two or three populations.